N-bromosuccinimide oxidation of a glucoamylase from Aspergillus saitoi.
1. In order to elucidate the structure-function relation of a glucoamylase [EC 3.2.1.3, alpha-D-(1 leads to 4) glucan glucohydrolase] from Aspergillus saitoi (Gluc M1), the reaction of Gluc M1 with NBS was studied. 2. The tryptophan residues in Glu M1 were oxidized at various NBS/Gluc M1 ratios. The enzymatic activity decreased to about 80% of that of the native Gluc M1 with the oxidation of the first 2 tryptophan residues. The oxidation of these 2 tryptophan residues occurred within 0.2-0.5 s. On further oxidation of ca. 4-5 more tryptophan residues of Glu M1, the enzymatic activity of Gluc M1 decreased to almost zero (NBS/Gluc M1 = 20). Thus, the most essential tryptophan residue(s) is amongst these 4-5 tryptophan residues. 3. 7.5 tryptophan residues were found to be eventually oxidized with increasing concentrations of NBS up to NBS/Gluc M1 = 50. This value is comparable to the number of tryptophan residues which are located on the surface of the enzyme as judged from the solvent perturbation difference spectrum with ethylene glycol as perturbant. 4. In the presence of 10% soluble starch, about 5 tryptophan residues in Gluc M1 were oxidized at an NBS/Gluc M1 ratio of 20. The remaining activity of Glu M1 at this stage of oxidation was about 76%. On further oxidation, after removal of soluble starch, the enzymatic activity decreased to zero with the concomitant oxidation of 2 tryptophan residues. The results indicated that the essential tryptophan residue(s) is amongst these 2 tryptophans. 5. The UV difference spectrum induced by addition of maltose and maltitol to Gluc M1 showed 4 troughs at 281, 289, 297, and 303 nm. The latter 3 troughs were probably due to tryptophan residues of Gluc M1 and decreased with NBS oxidation.
D001230:Aspergillus / Q000201:enzymology*; D001974:Bromosuccinimide / Q000494:pharmacology*; D055598:Chemical Phenomena; D002621:Chemistry; D005087:Glucan 1,4-alpha-Glucosidase / Q000302:isolation & purification; D005959:Glucosidases / Q000302:isolation & purification; D010084:Oxidation-Reduction / Q000187:drug effects; D013053:Spectrophotometry / Q000379:methods; D013329:Structure-Activity Relationship; D013388:Succinimides / Q000494:pharmacology*; D014364:Tryptophan / Q000032:analysis
D013388:Succinimides; D014364:Tryptophan; D005959:Glucosidases; D005087:Glucan 1,4-alpha-Glucosidase; D001974:Bromosuccinimide